Chronic orofacial pain is a common clinical syndrome lacking specific and effective therapeutic agents due to the fact that cellular mechanisms of chronic orofacial pain are poorly understood. Based on our preliminary data from a trigeminal nerve injury model and in non-orofacial pain models, we hypothesize that trigeminal nerve injury induced thrombospondin-4 (TSP4) expression in trigeminal ganglia (TG) and associated brainstem/upper cervical spinal cord (Vc/C2) that causes sensory neuron hyperexcitability, and abnormal synaptogenesis in the trigeminal complex in the spinal cord. These changes underlie the transition from trigeminal nerve injury to chronic pain development. In this proposal, we plan to identify the critical domain(s) of TSP4 in mediating behavioral hypersensitivity and spinal neuron hyperexcitability. Viral driven TSP4 expression in TG or Vc/C2, respectively, will be used to identify the site of the TSP4's action in chronic pain processing. We will perform confocal and electron microscopy to determine the extent of abnormal synaptogenesis in the nerve injury models. In addition, the influence of descending modulatory pathways and voltage-gated-calcium channels on TSP4-mediated behavioral hypersensitivity and dorsal horn neuron hyperexcitability will be studies using respective drugs. The influence of TSP4 on sensory neuron excitability, calcium channel activities, and intracellular calcium signaling will be studied in isolated neurons or intact TG from nerve injury models, or after TSP4 treatment. To determine if TSP4 induces behavioral hypersensitivity and dorsal horn neuron hyperexcitability through its interactions with its receptor, the calcium channel alpha-2-delta-1 subunit (Cava2d1), in a sensory neuron specific manner, Cava2d1 conditional knockout mice with selective deletion of Cava2d1 in subpopulation of sensory neurons will be used for these studies. The final goal of the proposed studies is to identify the peripheral and/or central mechanisms underlying TSP4-mediated transition to chronic pain states after trigeminal nerve injury. PUBLIC HEALTH RELEVANCE: Chronic orofacial neuropathic pain often evolves from a preceding injury of the peripheral nerves, which is accompanied by initial nociceptive pain. Identification of the mechanisms underlying this transition would be critically valuable in preventing or reversing it. We plan to study a novel pathway mediated by injury-induced expression of thrombospondin 4 in mediating orofacial neuropathic pain. Completion of this study will provide important information for identifying a novel mediator for the transition to chronic orofacial pain after nerve injury.